9 22 Exam II: Study Guide

Exam II Study Guide Lecture 3, cont—Leukocyte Migration and Inflammation Inflammation—3 types: Acute, systemic, and chronic   Objectives of inflammation: o 1. To destroy all injurious agents and to remove them from the inflammatory site o 2. To wall off and confine these agents to limit their effects on the host o 3. To stimulate and enhance the immune system o 4. To promote healing Acute inflammation—the one already discussed in study guide 1 o Overall effects:  Blood vessels dilate → increased blood flow to the area  Vascular permeability increases → outward leakage of plasma → exudate → edema  White blood cells (WBCs) adhere to the inner wall of blood vessels and then migrate through the walls to the site of inflammation o Process—acute inflammation begins within seconds after the injury  1. Initially, vasoconstriction happens, but briefly  2. This is followed by arterial vasodilation, which leads to increased pressure to allow…..  3. Exudation of plasma and blood cells into the tissues, which leads to edema and swelling  4. As plasma leaves, blood becomes more viscous and WBCs adhere to the walls of vessels  5. Leukocytes penetrate cell walls and enter the tissues  First, are phagocytic and non-specific killer cells  Neutrophils first, and then macrophages o Histamine is released to cause smooth muscle contraction and vasodilation  Anti-histamines prevent contraction and reduce swelling o Prostaglandins lead to vasodilation and increased vascular permeability  NSAIDs, such as aspirin and cox-2 inhibitors inhibit this and decrease pain o Macrophages secrete three cytokines that induce localized and system changes in acute inflammation  These are called pro-inflammatory cytokines  1. IL-1  2. IL-6  3. TNF-α (Tumor necrosis factor)  Locally, these act on fibroblasts and endothelial cells  Increase vascular permeability 1 Increase expression of adhesion molecules on cell surfaces Increase synthesis of chemokines that are chemotactic factors for neutrophils  As we discussed before, and will again later, many other cytokines then activate and modulate the function of all immune system cells o TGF-β (transforming growth factor-β) acts to limit inflammatory response so that healing can begin  This is secreted by Treg cells during suppression  Also secreted by macrophages, lymphocytes, and platelets at certain times o We can get local inflammation without involving the adaptive immune system, but, with time, the chemokines and cytokines usually attract lymphocytes  Therefore, we also have immune involvement Systemic acute phase response o When acute inflammation (above) is prolonged or extensive, it can be accompanied by a systemic response called the acute phase response, due to rapid alteration in the levels of several plasma proteins o Characterized by:  1. Fever—IL-1 acts on the hypothalamus and causes increased body temperature  This limits growth of some microorganisms  Also enhances the function of some immune system cells  2. Increased synthesis of hormones like ACTH and hydrocortisone  3. Increased white blood cell production in the bone marrow  4. Production of large numbers of hepatocyte-derived proteins such as….  Serum amyloid A protein (SAA)  C reactive protein (CRP) increases by 1000 fold in the serum o CRP binds to microorganisms and activates the complement system, which deposits C3b on the organisms o C3b leads to better phagocytosis (opsinization)    2 o Figure 13-14—IL-1, IL-6, and TNF-α produced by activated macrophages at the site of inflammation are particularly important in mediating acute-phase effects endothelial cells IL-1, TNF alpha IL-6 M-CSF G-CSF GM-CSF Liver hematopoiesis increased WBCs hypothalamus Acute phase proteins CRP SAA fibrinogen ACTH prostaglandins adrenal cortex fever corticosteroids  Chronic inflammatory response—chronic inflammation develops when there is prolonged exposure to an antigen o Examples include…  Microorganisms that are resistant to phagocytosis and thus remain around  Autoimmune diseases where self-antigens continually activate T cells  Rheumatoid arthritis  Relapsing multiple sclerosis  Tumors where chronic inflammation leads to tissue damage and wasting with some cancers o Accumulation of macrophages in chronic inflammation can stimulate other cells to increase collagen production, which leads to fibrosis at the site  Lots of activated macrophages surrounded by lots of lymphocytes is called a granuloma  Granulomas palpate like tumors—masses o Two important cytokines in this process are interferon-γ (IFN-γ) and TNF-α  In particular, TNF-α is responsible for wasting (called cachexia) common to some cases of chronic inflammation with certain microorganisms, parasites, and tumors  Later, we will discuss cancer and the immune system because TNF-α is used in cancer therapy 3  Anti-inflammatory agents o Inflammation is good…. Until it is not o Unwanted inflammation:  Allergies  Autoimmune diseases  Some prolonged microbial infections  Transplants  Burns  All of these can progress to chronic inflammation  We would like to reduce the inflammatory effects in these cases o Willow bark—the earliest used anti-inflammatory agent—aspirin o 1. NSAIDs (aspirin and cox-2 inhibitors)  Inhibit production of prostaglandins  Limit the increase in vascular permeability  Limit neutrophil chemotaxis in the inflammatory response  NSAIDs are effective for many chronic inflammatory diseases and some acute ones  Side effects of these drugs include GI difficulties  Danger of Reye’s syndrome in children with viruses who are given aspirin  This is hard on the brain and liver and can induce coma  Risk when child has chickenpox, the flu, mumps, measles, and others o 2. Corticosteroids—cholesterol derivatives (prednisone and others) that are potent anti-inflammatory agents  Decrease circulating lymphocytes  In humans, mostly thought to change migration patterns of lymphocytes  Reduce phagocytic and killing ability of macrophages and neutrophils  Reduce chemotaxis so fewer cells are attracted to the site o 3. Most modern drugs in use are antagonists of TNF-α such as Enbrel and Remicade  These work on long-term inflammation 4 Lecture 4—Antigens, Immunogens, and Epitopes (Beginning of Acquired Immunity)     Antigens are substances capable of inducing an immune response o We understand these things in order to understand….  1. All immune-driven treatments and diseases  2. How some things can escape the immune system  3. Vaccines  4. Many other mysteries of life and medicine Immunologic properties of antigens (two main properties) o 1. Immunogenicity—ability to induce a humoral (Ab) and/or cell mediated immune response  Thus, an antigen can be an immunogen  B cells + Ag → effector B cells (B cells and plasma cells) + memory B cells  T cells + Ag → effector T cells (T helper cells and CTLs) + memory T cells o 2. Antigenicity—ability to combine specifically with antibodies or T cell antigen receptors (TCR)  All molecules that can be immunogens can function as antigens; however, the reverse is not true…. That is, all antigens cannot be immunogens  Some small molecules, called haptens, are capable of binding to Ab or TCR, but cannot elicit an immune response; thus, the haptens are not immunogenic Factors that influence immunogenicity: o For humoral immunity, proteins are maximally immunogenic and carbohydrates are less so o For cellular immunity, only proteins are immunogenic o Nucleic acids and lipids of an infectious agent are not immunogenic unless complexed to a protein or polysaccharide  However, recall PRRs/TLRs and direct binding to DNA, lipids, carbs, etc.  PRRs are pattern recognition receptors that allow macrophages to respond in a semi-specific manner to invading organisms  Receptors look for specific patterns and repeating patterns, as in DNA, RNA, sugars, and phospholipids  C-reactive protein—acute phase response  MBL—mannose binding lectin  TLRs—specific receptors associated with host cell (surface or inside)  Recognize patterns characteristic of macromolecules Contribution of the immunogen to immunogenicity (4 things) o 1. Foreignness—under normal circumstances, a molecule must be recognized as nonself by the immune system  In principle, recognition of self occurs during development of the immune cells 5 Any immature immune system cells that interact with self are eliminated  This is called tolerance and will be discussed later  Any molecule not reactive to immature immune cells is later recognized as non-self or foreign by the immune system  Non-self is open for attack and, therefore, is immunogenic  Examples:  1. BSA (bovine serum albumin) is not immunogenic in cattle, but is immunogenic in humans, so drug companies cannot formulate insulin in BSA o Instead, they require HSA o If BSA is used, you get an immune response when you inject the insulin  2. Degree of immunogenicity is usually related to the phylogenetic distance o For instance, BSA will be less immunogenic in a goat or a human than in a chicken because goats and humans are closer phylogentically to pigs than are chickens  3. Corneal tissue and sperm occur in such immunologically privileged sites that they can be immunogenic in the animal that produces them o 2. Molecular size—in general, the bigger the protein, the better the immunogen  Thus, proteins with MW around 100,000 are usually excellent immunogens and proteins between 5,000 and 10,000 are not usually very immunogenic  < 1000 is thought to be too small to be immunogenic; however, some proteins with MW less than 1000 are highly immunogenic  Therefore, this is not a fixed rule o 3. Chemical composition and heterogeneity—size and foreignness are not enough  A synthetic polymer of only one amino acid is not immunogenic, regardless of size  If you use 2 amino acids, you get some immunogenicity and the degree of immunogenicity depends on which amino acids you choose  So, increasing complexity decreases the need for size of the antigen  How does the complexity work?—Effects on protein structure:  Most especially important for B cell antigen receptors (Ab)….  All four levels of protein organization contribute to the structural complexity of the protein and, hence, to its immunogenicity o 4. Degradability—most especially important for T cell antigens  To activate the immune system, especially T helper cells and CTLs, you require antigens that have been processed and presented on MHC on the cell surface  6 This means that dendritic cells and macrophages must be able to degrade the antigen o So now we have discussed the contribution of the immunogen to immunogenicity  However, a protein can have all 4 characteristics and still not be immunogenic in a certain organism…… (see below) Contribution of the biological system to immunogenicity o 1. Genotype of the recipient animal—some people respond to some immunogens while others do not  Usually, this is mapped to differences in the MHC molecules that we each express  Example: use two genetically different strains of hillbilly mice (inbred) and inject each with the same immunogen and look for serum antibodies  Mouse 1 might have high amount of Ab, while mouse 2 has low amount  After a cross (breeding between the mice) the offspring had intermediate levels of antibody when injected with the antigen  After doing a lot of genetics, it was determined that MHC is responsible for the difference in immune responsiveness  So cross-bred mice had half of the mom and half of the dad MHC  Many alleles encode Class I and Class II MHC (HLA) molecules and we get an assortment from each parent  ***This is the basis of tissue matching with respect to foreign and self***  Thus, MHC plays a central role in determining immunogenicity in a specific animal, but all MHC do not present all antigens  The genes encoding antigen receptors (B and T cells) and other proteins also play a role—this is discussed later o 2. Role of immunogen dosage and route of administration—How does the biological system respond?  This is related to immunizations and to de-sensitization shots for allergies  Also related to the immune response in general  Dosage level  If the dose injected is too low, you get no immune response because…. o 1. Not enough lymphocytes get activated or… o 2. You get a state of non-responsiveness in the lymphocytes  If the does is too high, you can get no response because the lymphocytes enter a state of non-responsiveness  Non-responsiveness = tolerance o We will spend a lot of time on this later   7 Dosage number  With many immunogens, a single does will usually not induce a strong response  You need to either survive an infection or get repeated doses to get a strong response (the idea of booster shots)  These increase the probability of clonal proliferation of antigenspecific T or B cells o Lead to an increased secondary response and thus, an increased immune readiness  Route of administration—usually parenterally (not via the digestive tract)  In general, I.V., I.D (intradermal), subQ (subcutaneous), I.M (intramuscular), and I.P (intraperitoneal)  The route determines which immune organs and which cells are involved in the response o IV goes to the spleen first o SubQ goes to the local lymph nodes (draining nodes)  Differences in the lymphocytes in theses organs controls, to some degree, the quality of the immune response o Thus, for each new immunogen, you must determine does response curve and optimal route for getting peak immune responsiveness for the vaccine that you are designing o 3. Adjuvants—from the Latin word, adjuvare, which means “to help”  Substances that, when mixed with an antigen, will enhance the immunogenicity of the antigen  Adjuvants are big business now  Selection of the best one is crucial, especially for an aging population  Example: Inject influenza vaccine with and without adjuvant and get different titers  Titer is the reciprocal of the last dilution of the patient’s serum to give anti-viral activity o Effective dilution = 1/512; therefore the titer = 512  Without adjuvant, influenza virus titers are not as high as with adjuvant: Lowest titer peak titer highest titer Virus 32 64 1024 Virus + adjuvant 64 512 8192   Adjuvants used to boost immune response when…..  1. The antigen has low immunogenicity (i.e. it is too close to self)  2. The antigen is in small concentrations 8 3. You are a company and you want to stretch the antigen ($$$) as far as possible  4. Aging o Possible mechanisms for adjuvant function:  1. Prolongs antigen persistence—anything that sequesters the antigen and allows slow release into the animal  Usually insoluble salts of something  Classic adjuvant is Freund’s adjuvant….  Incomplete Freund’s adjuvant (IFA) is an antigen in solution + a mineral oil + emulsifying agent to get the oil into small droplets surrounding the antigen—allows for slow release of antigen  2. Induces inflammation and granuloma formation—some adjuvants stimulate a cellular infiltrate at the site of injection  This is a granuloma with dense cells, including mostly macrophages packed closely together  This dense cell group is thought to increase phagocytosis and, therefore, antigen presentation and increased IL-1, resulting in increased TH cell activation  Complete Freund’s adjuvant (CFA)—incomplete Freund’s adjuvant + heat killed mycobacterium o Cell wall components activate macrophages or dendritic cells, which lead to increased IL-1 production, which activates TH cells  3. Stimulation of antigen presenting cells—CpG, which stimulates cells through TLR 9 is being examined as an adjuvant  Epitopes    As we discussed, the immune system does not interact with an entire immunogen molecule o Instead, it sees (interacts with) discrete sites called epitopes or antigenic determinants Epitopes are the discrete, immunogenically active regions of an immunogen that interact with antigen receptors on membranes or with secreted antibodies in solution Overview: o T cells and B cells have fundamental specializations for recognizing antigens o B cells:  Interact with antigen via a binary complex of the antigen and the antibody  Bind soluble antigens  Have no involvement with MHC  Antigens include proteins, polysaccharides, lipoproteins, DNA (antigenic only) 9  Epitopes are surface-accessible, hydrophilic, mobile, and often non-sequential in the protein (conformational epitopes) o T cells:  Interact with antigen via a ternary complex of antigen, TCR, and MHC  Do not bind soluble antigens  MHC is necessary  Antigens are only proteins  Epitopes are internal, denatured, amphipathic, and linear peptides that bind to MHC—continuity of antigen in peptide chain o B cell epitopes are usually hydrophilic and available in solution  These are generally found at a bend of the protein, which allows them more mobility within the protein o T cell epitopes must be linear because they are processed into peptides and presented on MHC  Also must be a protein  Because the MHC is required, the TCR recognizes a combination antigen, made from two different proteins (MHC and peptide) Now, greater detail  Properties of B cell epitopes o 1. The size of a particular epitope is determined by the size and conformation of the antigen binding site on a particular antibody and all of these differ  Interactions between Ag and Ab are due to weak non-covalent interactions, like H bonds  This means they are reversible  Comparison: (figure 4-14)  1. Small peptide hormones, like angiotensin II(8 amino acids) bind into a relatively deep groove in their antibodies  2. Huge, globular proteins (lysozyme), tertiary structure involving up to 22 amino acids can be involved and bind to a more planar face of considerable size on the antibody  So antibodies are selected to bind to the appropriate targets o 2. B cell epitopes in native proteins are generally hydrophilic amino acids on the protein surface and are topographically accessible to the antibody  If the epitope is buried in the protein, the antibody could not get to it o 3. B cell epitopes can contain sequential amino acids or non-sequential amino acids  Sequential amino acids are easy to understand  An amino acid sequence folds and sticks out into solution where the antibody can bind to it 10 Non-sequential epitopes are formed when the protein folds and two areas interact by hydrogen bonding or other non-covalent forces  These are called conformational determinants  These are brought together by folding of the protein  One characteristic of this is that the antibody only binds to this type of epitope with the protein in its native state o 1. If you denature the protein, the epitope is gone o 2. If you fragment the protein (with enzymes) the epitope is gone or less well conformed o 3. If you chemically reduce the protein when it is normally held together by disulfide bonds, it can denature and lose the epitope o Other ways to denature are heat, surface tension, salt, pH and drying  Consider isolation of a protein and purification to make a vaccine o If your most important epitope is a discontinuous type, and you get even a little denaturation, you could lose immunogenicity o This would cause you to need to add more immunogen o You can denature a protein with bubbles that result from shaking it up  In diagnosis of HIV, you look at the patient’s serum and ask whether an Ab against HIV in present o In designing the test, you have to apply an HIV protein to a solid substrate and ask if the serum can react with it o However, binding to plastic can cause denaturation of the protein o You must be careful to avoid denaturation of the HIV protein during application or the assay will be less effective, which might lead to a false negative or, possibly, a false positive o 4. B cell epitopes tend to be located in flexible regions of the immunogen and display site mobility  If they have maximum flexibility within their structure, epitopes can better conform to an Ab binding site  Structural rearrangement that allows for a nice fit is aided by the fact that this is happening in aqueous solution o 5. Dominant epitopes—some epitopes elicit more pronounced immune response in a particular animal than other epitopes of the same protein  These epitopes are considered to be immunodominant  Immunodominance is thought to be due to both, the intrinsic properties of the protein and the animal’s particular biology  11  The subset of epitopes chosen by an animal to be immunogenic differs according to the species of the responding animal  Also differe from individual to individual of a single species, which can be a problem in a vaccine design  Immunodominant in one person is not necessarily immunodominant in another person due to host factors  Vaccines effect the middle group in a normal distribution, and the other people are protected by the fact that the majority has immunity Five properties of T cell epitopes  For a long time, there was a question about the type of antigens that T and B cells respond to: Secondary immune response Primary immunization      Secondary immunization Ab production Cell-mediated Native protein Native protein + + Native protein Denatured protein – + Because the epitopes are melted in the bottom case, only T cells, which have them degraded before recognition, can respond to the antigens The dilemma was that a cell-mediated response was induced by a denatured protein even when the first injection was a native protein o It was hard to figure out how the epitopes would work In 1987, it was found that proteins are denatured to be presented by MHC o Thus, the cell-mediated response is always to denature proteins even if a native protein has been administered o Therefore, destruction of the protein conformation does not affect its T cell immunogenicity 1. Antigenic peptides recognized by T cells form trimolecular complexes with the TCR and MHC o So antigens recognized by T cells need two interacting surfaces:  1. The epitope to interact with TCR  2. The agretope to interact with MHC  Note that TCR also binds with MHC o The binding cleft of MHC does not have the same binding specificity as TCR or Ab, and one MHC can usually bind many peptides 2. Oligomeric peptides function as T cell epitopes o If the peptide is too short, it will not bind properly to MHC o Two types of MHC:  Class I on all cells presents antigens to CD8(+) cytotoxic cells 12 The cleft of Class I MHC binds best to peptides with 9 aa, and less well to those with 8 – 11 aa  Sends out a signal that says “kill me!”  Class II on APCs presents antigens to CD4(+) helper cells  The cleft of Class II MHC binds best to 12 – 25 aa (17 is most common)  Sends out a signal that slows down the killing reaction 3. Antigen processing is required to generate peptides that interact directly with MHC o Recall that the antigen must be degraded in the APC so that the peptides can associate with MHC and be presented 4. Antigens recognized by T cells are often internal in the protein o Thus, the epitopes are exposed only during degradation/processing for the MHC 5. Immunodominant T cell epitopes are determined in part by the set of MHC expressed by an individual o In a particular individual, the peptide that best binds to that person’s MHC also usually gives the best T cell immune response o So immunodominant for person A is not always the same as immunodominant for person B in T cell epitopes as well     Haptens and Antigenicity    Recall that haptens are not immunogenic, but do react with existing immune receptors o Drugs, peptide hormones, and steroid hormones can be haptens If you conjugate haptens to large carrier molecules, haptens can become immunogenic o These are called hapten-carrier conjugates o This is used to make antibodies for research and sometimes for commercial uses, such as diagnosis or therapy o But, this can also make drugs immunogenic when they form adducts with normal molecules and no longer function as haptens The original home pregnancy kit used the property of haptens o HCG is a hapten found in the urine of pregnant women o You can make a conjugate with a large particle and also get an antibody o When the antibody binds to the particles, it cross-links them and you see aggregation (clumping)  If the conjugate particles are blue, you see formation of blue color o Now, mix this reaction with urine and if the urine contains HCG, the HCG will bind to the Ab and block the clumping of the conjugate HCG  Therefore, when the person is pregnant there is no blue color 13 Mitogens     Recall that lymphocytes first proliferate to function by clonal expansion in response to a specific antigen Mitogens are agents that induce cell division (mitogenesis) in a high percentage of T and B cells regardless of the antigenic specificity of the cells o Because they do not care about specificity (clonality) of the cells, these are called polyclonal activators (mimic antigens) o These are worth knowing because they are used in research and in determining things about various cell types  It is important to know since some (PHA) are used in hospitals in diagnosis and some cause disease o Many are sugar-binding proteins (called lectins) from plants  Lectins bind sugars, therefore, lectins will cross-link glycoproteins on the cell surface, which can induce proliferation o Some preferentially activate T cells and some preferentially activate B cells and some do both  Examples: PHA = phytohemagglutinin—from kidney beans (activates T cells)  Hospital diagnosis for immunodeficiency: Do Pt cells proliferate in response to PHA?  Finds out if T cell response is still functioning  Boil kidney beans to deactivate PHA  Not boiling gives a GI response similar to food poisoning Bacterial antigens o Lipopolysaccharide (LPS) component of the gram-negative bacterial cell wall is a B cell and macrophage mitogen  LPS is mitogenic due to its lipid moiety, which interacts with the TLR-4 on the plasma membrane and leads to activation of the host cell  LPS stimulates macrophages to activate, proliferate, phagocytize and secrete stuff (inflammation)  LPS also stimulates B cells to proliferate  LPS is used in research to do this  In biotechnology, this is a big problem****? Superantigens—among the most potent T cell mitogens o Come from bacteria and viruses o Bind to one subunit of the TCR (β subunit) and also bind to Class II MHC o In both cases, the super antigen does not bind to the antigen-binding site of the TCR or MHC 14 o So the superantigen cross-links the TCR-MHC and mimics antigen stimulation even though the peptide (antigen) is not specific for the TCR and the cells proliferate and make cytokines o Interestingly, all superantigens do not activate all T cells  So you get a subset of T cells activated  Estimates that 1 out of 5 cells get activated by 1 superantigen o Examples:  TSST-1—toxic shock toxin produced by Staphylococcus aureus  Activates a large number of TH cells and gets massive release of cytokines (cytokine storm), which have systemic effects that contribute to pathology  Causes toxic shock syndrome  Can lead to death even after the bacterium is dead 15 Lecture 5—Immunoglobulins: Structure and Function     Immunoglobulins function as antibodies o On the surface of cells, they are antigen receptors  When the antigen binds, B cells proliferate and make more Ab o Secreted, they enter the blood, tears, saliva, etc. and are effectors of humoral immunity All immunoglobulins…. o 1. Bind to antigens o 2. Participate in effector functions How do they work? How are membrane bound and secreted immunoglobulins different? Immunoglobulins are good models for T cell antigen receptors (TCRs) Basic structure of immunoglobulins  First, why do they call it “i.v. gamma globulin” in the hospital and on TV?? o Old experiment: Put serum in electrophoresis and you see four peaks  Abumin peak, α peak, β peak, and γ peak  You know the antibody is somewhere in the serum, but which peak is the Ab?  At this time, proteins were called globulins Globulins Albumin    electrophoretic migration     The light line is the normal serum not treated with ovalbumin  Antibodies present in γ region Made anti-serum in rabbits against chicken Ovalbumen and then mixed that serum with ovalbumin and the antibody precipitated out  What was left was the dark line, which was known to be without antibody The conclusion was that the γ globulin fraction had the antibody in it and that the others did not Since then, the antibody fraction of serum is called the γ globulin  This contains all types of antibody 16 The antibodies in serum were called immunoglobulins to distinguish them from other proteins (globulins) in the γ fraction Antibody structure o Basic structure has two heavy and two light chains that are held together by disulfide bridges  The same structural model is true of the T cell receptor o Each antibody can bind two antigens, no more.   NH3+ NH3+ NH3+ NH3+ heavy chain light chain S S S S S Fab S Fab papain S S SS pepsin S S NH3 + S S F(Ab') 2 COO- COO- S S S S SS o Above is a prototype structure of IgG, showing chain structure and interchain disulfide bonds  The fragments shown are those produced by enzymatic digestion with pepsin or papain  Papain fragments:  The Fab fragments (45,000 Da each) have antigen binding activity  The Fc fragement has no antigen binding activity at all (right side), and because it was found to crystallize during cold storage, it was called Fc  Pepsin fragments:  The F(ab’)2 is a single fragment of 100,000 Da that is composed of two Fab-like subunits o This fragment binds antigens 17   Fc fragment is not discovered in pepsin treatment because it is digested into numerous small peptides Multiple myeloma o Plasma cells make boatloads of Ab o When they are transformed to tumors, they are called myeloma cells and they may continue to make antibodies  These antibodies are now called myeloma protein (so that you know they’re tumor-derived) o Myeloma protein can be as much as 95% of the serum immunoglobulins in patients with multiple myeloma o Some patients also secrete high levels of excess light chain into their urine  This was called Bence-Jones protein, after the discoverer  This was used to identify the nature of the antibody molecule, since before this they couldn’t get enough protein to determine the amino acid sequence o It was found that 100 to 110 amino acids at the amino terminal were different in each patient’s light chain o Also found that the rest of the amino acids formed into two groups that were the same from patient to patient  These two groups were named kappa and lambda light chains constant aa sequence LIGHT  100 to 110 aa = variable CHA IN Figure 4-12—variability of amino acid residues in the light variable domain and the heavy variable domain of antibodies with different specificities  Three hypervariable regions (called complementarity-determining regions [CDRs]) are present in both heavy- and light-chain variable domains  The three hypervariable regions in the light chain variable domain are brought into proximity in the folded structure  The same is true of the heavy chain variable domain 18 o Myeloma proteins were also used to determine the heavy chain sequence of amino acids  Again, it was found that 100 – 110 amino acids were variable between different patients  This was named the variable region  The rest of the protein fell into five groups that didn’t vary among the groups  These groups were named μ, α, γ, δ, and ϵ  The groups were called constant regions o The class of each antibody is named according to the constant region of its heavy chain:  IgM = μ  IgG = γ  IgA = α  IgD = δ  IgE = ϵ  Each of these major subclasses is called an isotype o After more was learned, it was concluded that some minor differences allowed the heavy chains to fall into subgroups  So, there are two A chains (α1 and α2) and four G chains (γ1 – γ4) o Figure 4-6—schematic diagram of structure of immunoglobulins derived from amino acid sequence analysis  Each heavy and light chain in an immunoglobulin molecule contains an amino terminal variable region that consists of 100 – 110 aa and differes from one antibody to the next  The remainder of each chain in the molecule (the constant regions) exhuibit limited variation that defines the two light chain subtypes and the five heavy chain subclasses  Some heavy chains (γ, δ, and α) also contain a proline-rich hinge region  The amino terminal portions corresponding to the variable regions bind to the antigen  Effector functions are mediated by the carboxy terminal domains  The μ and ϵ heavy chains, which lack a hinge region, contain an additional domain in the middle of the molecule  CHO denotes a carbohydrate group linked to the heavy chain 19 o Figure 4-10  A) heavy and light chains are folded into domains, each containing about 110 amino acid residues and an intrachain disulfide bond that forms a loop of 60 amino acids  The amino terminal domains corresponding to the V regions bind to the antigen; effector functions are mediated by the other domains  B) the μ and ϵ heavy chains contain an additional domain that replaces the hinge antigen o Note:  The immunoglobulin domains bound by disulfide bridges  Hinge regions  Carbohydrate glycosylation sites  Ag binding sites  Sites for biological activity  Each heavy chain can interact with a kappa or a lambda light chain o Table 4-3: Class Heavy chain light chain subclasses IgG γ κ, λ γ1, γ2, γ3, γ4 IgA α κ, λ α1, α2 IgM μ κ, λ none IgD δ κ, λ none IgE ϵ κ, λ none 20 Immunoglobulin fine structure    Immunoglobulin (Ig) domains o See figures 4-11 and 4-11 (above) o Each Ig domain is ~100 amino acids o Each has one intrachain disulfide bond o Within the disulfide bond are ~60 amino acids o Each domain folds into a recognized structure called the Ig fold  Two parallel β sheets  Each sheet consists of 3 or 4 antiparallel β strands  Two sheets are joined by the disulfide bridge o Note: the loops between the β strands  These can be variable in length and, as we see later, can vary in sequence to form antigen binding areas Figure 4-9 shows two representations of an intact antibody molecule o Interactions between domains in the separate chains of the immunoglobulin molecule are critical to its quaternary structure o The different domains interact very nicely with each other to form an immunoglobulin molecule  For example, CH2 from on heavy chain interacts with CH2 on the other heavy chain  CH3 with CH3 and CH4 with CH4  Also, the heavy and light chains interact: CH1 with CL and VH with VL o Hypervariable regions come from the β turns Figure 4-12 (drawn above on p. 18) o Now, it is reasonable that if you want to change the ability of an Ab to bind to something, you don’t have to change all of the Ab, just the binding region  So, if you compare the amino acid sequence of several different antibodies raised against the same antigen, you can look for which amino acids are the same and which are different 21     o Study the sequence variability in Ab with different specificities  Note: in each Ab are areas of sequence variability called hypervariable regions or complementarity determining regions (CDR)  CDR-1, -2, -3 are complementary to Ag  The rest that do not change are called framework regions (FR) Each heavy chain has 3 CDRs and each light chain has 3 CDRs o These are loops between the β regions and they form the antigen binding site o The framework regions serve as scaffolding to align the Ag binding regions properly o Note that if you alter only one amino acid, you could alter the specificity o Interaction of Heavy and Light chains puts together the VL and VH and is the final event that determines antigen specificity Now, about the binding surfaces o If the antibody is binding a large, globular protein, like the neuraminidase of influenza, the antibody contacts the antigen on a flat, undulating surface and in the area of contact, protrussions and depressions of the antigen protein are matched by the antibody protein o In contrast, smaller antigens like the hapten phosphorylcholine or the octapeptide hormone, angiotensin II, are targeted by a smaller binding site with more of a deep pocket in which the antigen gets buried Constant region domains—structure and function o Constant region is responsible for various biological functions o The amino acids of these domains bind to Fc receptors  Recall, the Fc portion of the Ab is the back end, which is away from the variable region o Examples:  IgG and IgM antibodies use the Fc region to bind Fc receptors on phagocytic cells, which leads to opsonization  Some IgG subclasses bind to Fc receptors on placental cells and are then transferred across the placenta to protect the fetus as maternal antibodies  IgE uses Fc receptors to bind to mast cells and basophils  Mast cells will pick up random antibodies and stick them on  Therefore, the mast cell has a broader antigen response Immunoglobulin-receptor complex o Not all immunoglobulins are secreted o Some remain associated with the membrane to form the B cell antigen receptor  This is a different form of Ab o The surface Ab thus shows the antigen specificity of that particular B cell o Membrane-bound Ig differs from secreted Ig at the carboxy terminal end 22 o Each of the 5 isotypes can be expressed as membrane bound antibody Antigenic determinants on immunoglobulins     Three types: Isotypic, allotypic, and idiotypic 1. Isotypic determinants—an antigenic determinant (epitope) within the immunoglobulin constant region that is characteristic of a species (answer to the question of “what makes IgG different from IgM?”) o These are the constant region amino acid sequence antigen groups o Inject an antibody from one species (mouse) into a different species (human), and get an immune response to the foreign Ab and see human-anti-mouse Ig antibodies  This is why we cannot treat humans with mouse-derived Ab 2. Allotypic determinants—arise from differences in alleles in individuals of the same species o Humans have more than one allele of, say IgA2. o Each represents a difference of 4 or so amino acids o These are spread along the heavy chain o If you inject one human Ab into a different human who does not have the same allotype, you can get antibodies against the allotypic determinants o Two examples:  1. Get antibodies to allotypic determinants during blood transfusions  2. Mother can raise antibodies against fetal allotypic determinants that are gotten from the father 3. Idiotypic determinants o Idiotope = a single antigenic determinant in the variable domains of an antibody or Tcell receptor; also called idiotypic determinants  Generated by the unique amino acid sequence specified for each antigen o The VL and VH regions of the antibody function to bind the antigen, but they also can be antigenic determinants due to their variability o The combination of all six determinants is called an idiotype o These are predicted to be good for vaccines  Idiotype → anti-idiotype antibody  ****Inject the anti-idiotype Ab and get an Ab that looks like the original antigen**** o So, if you have low levels of a viral protein and it is too low to make a vaccine….  1. Inject the viral protein (original antigen) into a mouse and get an antibody that binds to the original antigen  2. Inject the antibody into something else and you get anti-antibody antibodies, which bind to idiotopes on the original antibody 23 o Since the original antibody uses variable regions (and therefore idiotopes) to bind the original viral antigen, and the anti-antibody uses its variable regions to bind to the idiotopes of the original antibody…. o You have generated a variable region idiotope that binds to anti-viral antibody, just like the original viral antigen does o Therefore, you can inject the anti-antibody and raise new antibodies against the viral antigen‼‼ (complex) Specific immunoglobulin isotypes      Isotype—an antibody class which is determined by the constant region sequence of the heavy chain o Five human isotypes: G, D, E, A, and M, which exhibit structural and functional differences o Also refers to the set of isotypic determinants that is carried by all members of a species Each isotype has its own distinctive heavy chain Each heavy chain constant region confers specific properties on the antibody IgG—most populous type of antibody o Most abundant in serum (~80% of total serum Ig) o IgG forms as a monomer of 2 heavy and 2 light chains o There are four subclasses of IgG number by decreasing amounts in human serum  IgG1 = 9 mg/mL  IgG2 = 3 mg/mL  IgG3 = 1 mg/mL  IgG4 = 0.5 mg/mL o Each is encoded by a different gene and all are 90 – 95% homologous, but the differences matter! o IgG1, IgG3 and IgG4 readily cross the placenta and protect the fetus o IgG1 and IgG3 activate complement; IgG2 does, but not so well; and IgG4 doesn’t at all o IgG functions as opsonin and binds to Fc receptors (IgG1 and IgG3 are the best) IgM o The first antibody made in the primary immune response  Primary response is almost entirely IgM  Most secondary response is IgG o 5 – 10% of the total serum Ig—average concentration = 1.5 mg/mL o On the B cell membrane it is a monomer o Secreted as a pentamer 24  5 monomers held together by disulfide bridges plus one J chain disulfide bound to 2 of the monomers o See figure 4-17  Note that IgM and IgE heavy chains contain five domains and no hinge region, while IgA, IgG and IgD contain four domains and a hinge region  Polymeric forms of IgM and IgA contain a polypeptide, called the J chain that is linked by two disulfide bonds to the Fc region in two different monomers   On IgM….  The Fc is in the center and the antigen binds sites (10) on the outside  Increased valency per molecule makes IgM better at neutralizing viruses than IgG o IgG has better binding affinity, but IgM holds with ten arms, while IgG holds with two, so IgM is better  IgM is better at activating complement than IgG  IgM does not diffuse well into tissues because of its size o However, J chain allows it to bind to secretory cells, and to be transferred across epithelial cell linings to secretions that bathe mucosal linings  Not as much as IgA, but still some IgA o Only 10 – 15% total Ig in serum is IgA, but it is very high in mucosa 25 o It is the predominant Ig class in external secretions, such as breast milk, saliva, tears, bronchial mucus, genitourinary tract, and digestive tract o See figures 4-17 (above) and 4-19 (below) In serum, IgA is mostly a monomer Secretory IgA consists of two IgA molecules which are covalently linked together through a J chain and are also covalently linked to a polypeptide chain called a secretory component (~70,000 MW) produced by epithelial cells of mucus membranes  The secretory component has 5 Ig-like domains that bind to Fc regions of the IgA dimer Each day, humans secrete 5 – 15 grams of IgA into the mucus secretions Plasma cells that make IgA hone to the sub-epithelial tissue IgA binds to a receptor on the epithelial cell surface called the poly-Ig receptor  Then, gets internalized into vesicles, transported through the cell and the vesicle fuses with the plasma membrane  Along the way, the Ig receptor is degraded, leaving the secretory component bond to the IgA, which is ejected into the mucosa or other fluid The secretory component masks protease sites on the IgA, protecting it from proteases and leading to longer half-life in the intestines and lungs See figure 4-19 (above)  In mucosal secretions, IgA binds to bacteria and viruses and prevents them from attaching to the epithelial cells and thus inhibits growth and infection  These complexes of IgA and antigen are eliminated in the mucus  IgA is effective against Salmonella, Vibrio cholerae, Neisseria gonorrhea and viruses (influenza, polio and retrovirus) Also secreted into breast milk to protect newborns   o o o o o o  IgE o Very low average serum concentration (~0.3 μg/mL) but very high in the tissues 26  o Is thought to be important for defense against parasites o Mediate immediate type hypersensitivity reactions like hay fever, asthma, hives and anaphylactic shock o Named IgE after the E antigen of ragweed pollen, which is a potent inducer of IgE o IgE binds Fc receptors on the surface of mast cells and basophils o Antigens are allergens, which lead to cross-linking of IgE antibodies, which leads to degranulation of cells releasing lots of pharmacologically active mediators, which leads to manifestation allergic symptoms IgD (little dork) o Represents only 0.2% of antibody serum at 30 μg/mL o Biological function is not known o Thought to function in B cell activation, sometimes co-expressed with IgM The immunoglobulin superfamily     All heavy and light chain Ig have the Ig fold domain (figure 4-9, 4-10) at ~110 amino acids long o It is thought that Ig arose from single primordial gene encoding protein of 110 amino acids o Then, gene duplication and divergence gave rise to lots of proteins with groups of the Ig domain o We have found that mnay proteins have one or more domains like the Ig domain o See figure 4-23 for some examples, but do not memorize These are some, but not all of the members of the group that shares the Ig domain. o Members of the group are said to be members of the Ig superfamily We will encounter these in future lectures: o 1. All contain ~110 amino acids arranged with antiparallel β strands into β pleated sheets o 2. All have an intrachain disulfide bridge that captures 50 – 70 residues o 3. Not all have an antigen binding domain like immunoglobulins and TCR So one question is…. Why is the Ig domain conserved in evolution? o Theory:  Recall that constant region Ig domains in antibody interact to help formation of the heavy chain dimer  Perhaps, Ig domains help interaction of other members of the Ig superfamily  Either dimerization or counter-receptor recognition  Ig superfamily will be important throughout the course 27 Lecture 6—Antigen-Antibody Interactions   Antigen-antibody interactions: o 1. Bimolecular interaction (similar to an enzyme-substrate interaction) o 2. Reversible: NO irreversible chemical alteration of the antibody or the antigen, which is unlike an enzyme-substrate interaction o 3. Non-covalent interactions between the epitope and the antibody variable region (VLVH) = hypervariable regions = CDRs (complimentarity determining regions) o 4. Extremely specific In addition to protecting us, antibodies are used for…. o 1. Diagnosing diseases o 2. Monitoring the level of humoral response in a patient o 3. Identifying molecules of biological or medical interest o 4. Research o 5. Home self-determination kits, like pregnancy and HIV o 6. Therapy Strength of antigen-antibody interactions     1. Types of bonds and interactions: o Weak interactions (hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waal’s interactions) 2. In addition, the bond distance for each is comparatively short so you require a very close fit between the two proteins—reflected in the high degree of specificity 3. Beccause each is comparatively weak when compared to covalent bonds, you require a large number of individual bonds to hold the two protein stogether 4. Each bond is in an “on-off” equilibrium 1. Strength of the bond is measured by affinity for the antigen (Antibody affinity)  The strength of the sum total of all the weak interactions holding the antibody and the epitope together is the affinity of the antibody o Low affinity antibodies bind weakly and tend to dissociate more readily  These have a lower sum of total weak interactions or poorer fit o High affinity antibodies bind antigens strongly and remain bound longer o This knowledge is useful for designing diagnostic kits 28 The on rate may be slow and low but if the off rate is very low, you can still have a good antibody Assay—can measure the affinity by equilibrium dialysis o Not many immunologists do this anymore, but biotech people use it for comparing antibodies for designing kits, therapy and diagnostics    o See figure 6-2:  Dialysis chamber contains two compartments (A and B) separated by a semipermeable membrane  Antibody in A and radiolabeled ligand in B  At equilibrium, the amount of radioactivity in each compartment is measured  When no antibody in A, radioactivity is the same in each compartment, as it distributes itself evenly  When antibody is present, A will have more radioactivity than B  The higher the affinity of the antibody, the more of the ligand is bound at equilibrium  We can calculate the affinity constants and assign a value to each Ab for comparison to design your kit, therapeutic or diagnostic  Haptens can be used with this as well So, affinity is the overall strength of binding 2. Strength of binding measured by avidity for the antigen (Antibody Avidity)    If you have a multivalent antigen and you mix it with an antibody, interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site In a biological system (viruses and bacteria), the avidity is a better measure of the binding capacity of the antibody than is the affinity of the antibody Avidity can compensate for low affinity o Example: secreted pentameric IgM often has lower affinity than IgG, but the multivalence gives it high avidity and it binds a multivalent Ag effectively o Think off holding on with 10 hands, rather than just 2 3. Cross reactivity 29   Even though antigen-antibody reactions are highly specific, there are still some cases when the antibody elicited by one antigen can cross-react with the antibody elicited by a different antigen o 1. If two antigens share a common amino acid sequence o 2. If the antibody against one epitope reacts with a second epitope with similar chemical properties, such as similar charges and located in a similar place as the original epitope, but chemistry like different amino acids or carbohydrates, which can be similar o Usually, the antibody’s affinity for the second antigen is less than for its original antigen because it is usually not an exact fit Three examples: o 1. Vaccinia virus—the most famous is that vaccinia virus (cowpox) raises antibodies that react with variola virus (smallpox) o 2. Rheumatic fever and Bright’s disease—make antibodies against Streptococcus pyogenes during an infection with that bacterium  In some people, these antibodies can cross-react with the patient’s tissues  If the antibody cross-reacts with heart muscle, you have rheumatic fever  If the antibody cross-reacts with the kidney, you have Bright’s disease  The next time the patient gets an infection with S. pyrogenes, the protective antibodies attack the heart or kidneys  Thought to be similar with Chlamydia and atherosclerosis o 3. ABO blood groups—blood group antigens expressed on RBCs  People who do not express a particular antigen have antibodies against that antigen instead  For example, if you have Type A blood, you express an A antigen on your RBCs and make antibodies against the B antigen  If you have Type O blood, you don’t express either antigen and you have antibodies against both antigens  Cross reactivity does not come from the blood groups, since people who have never had a transfusion still express these antibodies  How does it work?  Say you are type A—you make antibodies against cross reacting microbial antigens present on intestinal organisms  These are anti-B antibodies since the anti-microbe antibodies cross react with B  But why don’t you make cross reacting antibodies against A?? o You do not make antibodies against the antigens that you already express since you have tolerized yourself to those antigens as a fetus 30 o Thus, you can’t make anti-A antibodies since you are tolerant to A o This is why O people make both! They aren’t tolerant to either as a fetus 4. Antigen-Antibody reactions     Antigen-antibody reactions in solution form a lattice of antibody and antigen that mimics immune complex diseases In solution, the initial antigen-antibody reaction forms in minutes o However, over much longer time, we get a visible precipitate because adjacent antibodies form bonds with the same antigen and make a big lattice Antibody must be bivalent for this to happen o A precipitate will not form with Fab fragments Antigen must be bivalent or polyvalent 5. Agglutination reaction    Antigen binds antibody and we see visible clumping called agglutination Hemagglutination (HA) is what happens when you do the RBC blood typing test o Mix patient RBCs with known typing anti-sera on a slide o If the RBC expresses type A, the anti-A antibody will bind to RBCs and make them clump or agglutinate (hemagglutination because of the RBCs) o If the RBCs express both antigens, they will react with both, Anti-A and Anti-B, we know they are Type AB o If the RBCs are type O, they express neither A nor B and will react with neither antiserum Agglutination inhibition o 1. First example is the pregnancy test where the urine HCG inhibits agglutination of the blue beads by the antibodies o 2. Second example is drug testing (for cocaine or heroin) (picture to the left side)  Coat RBCs with drug to make a passive agglutination reaction using your known antibody  Antibody against the drug will agglutinate the coated RBCs  Mix the anti-drug antibodies with patient urine  If the drug is in the urine, the drug will act as a hapten and block antibody binding to the RBCs, which will lead to no clumping  No clumping = patient is drugged up 31   One problem is that some legal drugs and foods have the ability to cross react with the antibody So if you get a positive with immunological methods (quick), you must verify with a non-immunological method  In this chemical test, we see no cross reactivity, but it takes longer and is expensive  This is why we weed out the negatives first with the immunological test and then do the expensive one 6. Methods for understanding the rest of the course  Enzyme Linked Immunosorbent Assay (ELISA) o The most famous use is to test for HIV—stage 1 testing o 1. First, stick a known antigen onto a play  Known: HIV antigen  Then wash o 2. Next, add a patient’s serum to be tested. Then incubate o 3. Rinse away any protein that does not stick to the plate o 4. Then do the detection procedure to see if any of the patient’s serum antibody stuck to the known HIV antigen, indicating a positive HIV test for antibody in the serum o 5. Detect by incubating with goat antibody made against human Ig  Previously, you would have covalently linked an enzyme to the goat antihuman IgA b o 6. After incubation and rinsing, add the substrate for the enzyme  The substrate changes color and the level of the color gives the degree of positivity o Other types include sandwich ELISA and competitive ELISA, which we will not deal with here o Sometimes, ELISA for HIV can give ambiguous results and must be verified with a more direct method  Western Blotting (used after ELISA if results are unclear) o Run electrophoretic gel of several HIV proteins 32 o Transfer the proteins to a membrane and probe the membrane with the patient’s serum o Then detect the serum antibody in the same way as with ELISA, using goat antihuman Ig antibodies o The advantages of this method are that you can see specific sizes of the proteins and know that what you’re getting is not a false positive o RNA = northern blot; protein = western blot; DNA = Southern blot (Ed Southern)  Immunofluorescence o Many assays and home diagnostic kits boil down to just asking where the antibody is o Another way to tell where an antibody is, is to tag it with fluorescent dye like fluorescein or rhodamine o Fluorescent dyes absorb light of one wavelength and emit light at a different wavelength  Fluorescein absorbs at 490 (blue) and emits at 517 (yellow)  Rhodamine absorbs at 515 (yellow) and emits at 546 (red) o Direct immunofluorescence—so if you couple one of these dyes to the Fc portion of an antibody, you can tell where the Ab binds by shining light of the appropriate wavelength on it and measuring the amount of fluorescent light emitted o Indirect immunofluorescence—sometimes it is expensive to couple dye to all your antibodies, so you couple them to a secondary antibody, like goat anti-mouse IgG and then stain with the primary antibody like mouse anti-human CD4 antibody  The primary is not labeled  Next, treat with secondary Ab (goat anti-mouse) which is tagged and will bind only where the primary Ab bound and can be detected by fluorescence detection methods o Mostly done two ways:  1. Using a fluorescence microscope which has the ability to do these things  Can recognize which cells are positive for you antigen and where in the cell the antigen is  For example, on the membrane, in the cytoplasm, in the nucleus, etc.  Take pictures and evaluate using computer programs for intensity of staining  2. Use a flow cytometer (hospitals use or send work away) which uses laser excitation to detect various fluorescent dyes within a mixed cell population  Sends cells through a detector one cell at a time and measures intensity of the fluorescence for each cell and then gives you a plot representing a whole population  Used in hospitals for CD4/CD8 ratios to determine decrease in CD4 in HIV patients 33    Used to detect leukemia marker Used to detect T cell-B cell ratios for immune sufficiency or immunodeficiency Used heavily in research 34